Vitamin D is a fat-soluble vitamin. Only a small portion of the vitamin D in human body is derived from food (<10%), while 90% of the vitamin D required in vivo derives from the ultraviolet irradiation on the skin by the Sun. The primary form of vitamin D in human body is vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The major nutrition sources of vitamin D2 are some vegetables, yeast, and fungi foods. Vitamin D3 is formed as a result of the conversion of subcutaneous 7-dehydrocholesterol affected by the Sun.
Vitamin D2 and vitamin D3 derived from food sources or autosynthesis enters the liver via the blood circulation and are converted to 25-OH vitamin D2 and 25-OH vitamin D3 by 25-hydroxylation enzyme, respectively, and referred to collectively as 25-OH vitamin D. The content of 25-OH vitamin D in the serum or plasma can reflect the total amount of vitamin D from food intake and autosynthesis. Therefore, 25-OH vitamin D is a best indicator of vitamin D nutritional status.
25-hydroxyvitamin D is a main storage form of vitamin D and an essential nutrient for the metabolic activity in the human body. In addition to the effect on the bones in the traditional sense, 25-OH vitamin D may also have a non-skeletal effect impact on autoimmune disease, cardiovascular disease, diabetes, cancer, and so on. Studies have found that vitamin D deficiency can directly affect the expression of human genes which are associated with many diseases such as rheumatoid arthritis, diabetes, and the like.
At present, the clinical quantitative detection of vitamin D has become a routine item in tests and physical examination. Therefore, it is particularly important to establish a fast, simple, sensitive, accurate, and reliable method of vitamin D detection. Quantitative detection of 25-hydroxyvitamin D mainly includes physical and chemical assays and immunological methods. The application of physical and chemical assays using, e.g., liquid chromatography-mass spectrometry (LC-MS) is limited due to the expensive equipment, complex sample preparation, time-consuming and tedious process, to name a few. Immunological method is sensitive, rapid, simple and specific, and thus has now become the primary vitamin D assay. A mainstream products currently on the market for the detection of 25-hydroxyvitamin D is a detection kit produced by Diasorin, Italian, the detection method of which is implemented by using ABEI (N-4-aminobutyl-N ethylisoluminol)-labelled 25-hydroxyvitamin D antigen derivatives that compete with the antigens in the sample to bind with the 25-hydroxyvitamin D antibody coated on magnetic microspheres, and then adding an excitation substrate to produce a luminescent signal, enabling quantitative detection of 25-hydroxyvitamin D. Such operating mechanism is better illustrated in FIG. 1 (the specific operation steps and experimental conditions may be found in the manual of DiaSorin-VD). In the detection process, the stability of ABEI-labeled 25-hydroxyvitamin D antigen derivatives is the most important factor affecting the stability of detection. However, 25-hydroxyvitamin D antigen derivative has poor stability, with its activity weakened when exposed to light or heat. Therefore, the reagents in the above detection kit suffers from poor stability and vulnerable activity.
Thus, the instability of the 25-hydroxyvitamin D antigen derivative used in the immunological competition assay method leads to a short storage life for immunological detection agents for 25-hydroxyvitamin D detection, which adversely affects their use and detection results. In addition, stringent storage conditions have caused great inconvenience in storage and transportation. All of these have become the bottleneck for large-scale commercialized application of such methods.